LOCUS       sequence-71709-e        8289 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for Cre-lox cassette exchange of untagged gene sequences into the S.
            pombe urg1 locus.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8289)
  AUTHORS   Watson AT, Daigaku Y, Mohebi S, Etheridge TJ, Chahwan C, Murray JM, 
            Carr AM
  TITLE     Optimisation of the Schizosaccharomyces pombe urg1 Expression 
            System.
  JOURNAL   PLoS One. 2013 Dec 20;8(12):e83800. doi: 
            10.1371/journal.pone.0083800.
  PUBMED    24376751
REFERENCE   2  (bases 1 to 8289)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2013-12-20"; volume: "8(12):e83800. doi"; pages: " 
            10.1371/journal.pone.0083800"
COMMENT     Sequence Label: pAW8ENdeI
FEATURES             Location/Qualifiers
     source          1..8289
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      575..1354
                     /label=ars1
                     /note="Schizosaccharomyces pombe autonomously replicating 
                     sequence ars1"
     primer_bind     complement(1449..1468)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1568..1590
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1628..1646)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1714..1818
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1819..1887,1888..2679)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2037..2056)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2850..3438
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3339..3358
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3592..3609
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3726..3747
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3762..3767,3768..3785,3786..3792)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3800..3816
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3805..3827
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3824..3840
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3824..3840
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    complement(3859..3892)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4027..4044)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4027..4043)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4036..4058)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             6938..8032
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKADAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
ORIGIN
        1 caaaaagtta ggtgtaacag aaaatcatga aactatgatt tctaatttat atattggagg
       61 attttctcta aaaaaaaaaa tacaacaaat aaaaaacact caatgacctg accatttgat
      121 ggagtttaag tcaatacctt cttgaaccat ttcccataat ggtgaaagtt ccctcaagaa
      181 ttttactctg tcagaaacgg ccttaacgac gtagtcgatc cggacgatgg ccaaccttcc
      241 aattcattaa atcgagtcct ccatcagatg atttacaagg ttttgcatag aatccctaag
      301 taagagaagt tgttagtgat ttcttcaagt ttactaaaat ttacatacaa atggatgatc
      361 tcttctccat tgtttccttt cttcttgcaa acgcgtttta caaagagatg acatgctatt
      421 ttctgattat ttttttctat agttttctat tttgtacgtc cttgggagtc cgaaatgtaa
      481 aatcggggta tggttagtag cggcagcgtt cattatggta tataaaagat aaaaagtagg
      541 ggcaagcgag acagaatggg atacaagggc atcgtctata attatagcta aaaattgtat
      601 tttaatttgt attttttgta attttatttc tcattgtttt acttaaaatg aatgcgaatt
      661 agagaaaact tcaacgaaat gtcaaaataa gctcagcaaa atatacaatt ttagggaaag
      721 cgatcagcaa cttattctcc ggtgtagatt ctttttcttc ataaattcca acaatgtaac
      781 tgccaattga tttttctcta gatgctaata aattgaaact aatgaatata ataacacagg
      841 ttttgataaa ttgtaaactc ttactcaaaa aaagaatgtt aaaactaatt taaccttttt
      901 ataaaggctc atggcgttaa agaggatagt aaaaattcta caatttttta atttaatgtc
      961 acaattgtct tgcgaatata attctattaa ttttcgctaa ctgctgtttt agtactatta
     1021 ctttcaatat gccttatatg caactttaat tcatgaagaa tgcaatattt ttgcaatctt
     1081 tcttgtactg agcgcttttt atcatttagt actcattatt taatttttca gtaaagggca
     1141 ataaataaat ttgtagaaga tgcaatgtaa tctcctctat ccttttgctc atgtttatga
     1201 gtatacctag tctagaaagg cttgtattta aaatattatt caataaaaat tcacaatttt
     1261 tacgacatgt gctaccattc acttaacttc ctgattataa aattggttcg tttatactaa
     1321 ttacttaagt acctttaact aaacaaaatg cctatatata tattaattta caatgagtgt
     1381 cagataagtc actatgtccg agtggttaag gagttagact cgccatcgat ggtatggtgc
     1441 actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca
     1501 cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg
     1561 accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga
     1621 cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct
     1681 tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
     1741 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
     1801 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
     1861 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
     1921 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
     1981 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     2041 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     2101 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     2161 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     2221 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     2281 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     2341 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     2401 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     2461 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     2521 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     2581 cgtatcgtag ttatctacac gacggggagt caggccacta tggatgaacg aaatagacag
     2641 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     2701 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     2761 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     2821 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     2881 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     2941 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     3001 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     3061 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     3121 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     3181 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     3241 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     3301 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     3361 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     3421 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     3481 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     3541 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     3601 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     3661 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     3721 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg
     3781 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     3841 catgattacg ccaagcttat aacttcgtat agcatacatt atacgaagtt attgctagcc
     3901 gttttcttca attactaaat tagaactaat tcaatcatat gctggcatgc ggagagctcg
     3961 gagtcgacga gactagtact cccgggataa cttcgtatat ggtattatat acgaagttat
     4021 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     4081 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     4141 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
     4201 tctccttacg catctgtgcg gtatttcaca ccgcatacca tcgtccggat caatgaagta
     4261 gatgagttta ctacctgtat atactttttt ttttgtcaaa gtcactttta tggcatttcc
     4321 atgttgaagg attagccaca gcactaaatt ttcagcgtat gattgctttt aaatatttaa
     4381 ttttcatcgt tttttaatta atattcaaaa cgatttaatg ccttctacat ctgaagtcaa
     4441 gctcatagac tcgggttggg tagctaaaac tacatctaac acgaaggggt atagccttct
     4501 taaagtatac tcatgtataa acgttttggg ttcatatttt tgggccacgt ggtttataaa
     4561 aattcttaac tacaccactc ggtatcccgc acccgtctac gtttctacga tttcgagcta
     4621 atattgagta aagtgattag caaaaaaaat aaaagtactc gttgtcggag atcaagaatt
     4681 tttctattat ctcatctaaa ccactttcta aaagcgaaaa acaaaatcgt aatatgcagc
     4741 ttgaatgggc ttccatagtt tgaaagaaaa accctagcag tactggcaag ggagacattc
     4801 cttttacctg gctaatcgcc atcttccagc aggcgcacca ttgcccctgt ttcactatcc
     4861 aggttacgga tatagttcat gacaatattt acattggtcc agccaccagc ttgcatgatc
     4921 tccggtattg aaactccagc gcgggccata tctcgcgcgg ctccgacacg ggcactgtgt
     4981 ccagaccagg ccaggtatct ctgaccagag tcatccttag cgccgtaaat caatcgatga
     5041 gttgcttcaa aaatcccttc cagggcgcga gttgatagct ggctggtggc agatggcgcg
     5101 gcaacaccat tttttctgac ccggcaaaac aggtagttat tcggatcatc agctacacca
     5161 gagacggaaa tccatcgctc gaccagttta gttaccccca ggctaagtgc cttctctaca
     5221 cctgcggtgc taaccagcgt tttcgttctg ccaatatgga ttaacattct cccaccgtca
     5281 gtacgtgaga tatctttaac cctgatcctg gcaatttcgg ctatacgtaa cagggtgtta
     5341 taagcaatcc ccagaaatgc cagattacgt atatcctggc agcgatcgct attttccatg
     5401 agtgaacgaa cctggtcgaa atcagtgcgt tcgaacgcta gagcctgttt tgcacgttca
     5461 ccggcatcaa cgttttcttt tcggatccgc cgcataacca gtgaaacagc attgctgtca
     5521 cttggtcgtg gcagcccgga ccgacgatga agcatgttta gctggcccaa atgttgctgg
     5581 atagttttta ctgccagacc gcgcgcctga agatatagaa gataatcgcg aacatcttca
     5641 ggttctgcgg gaaaccattt ccggttattc aacttgcacc atgccgccca cgaccggcaa
     5701 acggacagaa gcattttcca ggtatgctca gaaaacgcct ggcgatccct gaacatgtcc
     5761 atcaggttct tgcgaacctc atcactcgtt gcatcgaccg gtaactttta gattaacatt
     5821 tgtttaaata tgaaattagg caatgtaaca tactgcaggc aaattttggt gtacggtaag
     5881 taaattggac ataagattta acaaagcgac tataagtcag aaagtgagaa tgagattgaa
     5941 ataattaatt caacttattc aattgatgat atgccaggat tcctcttcct ttatcttcaa
     6001 tgtttcgatt aacaggtcca ttatccggtt tttagtttcg ctgcacattg ccgaatgaca
     6061 atcggattcc tttaagctat gcttttgccc tgttttctct ccccagtaat cgttgggtga
     6121 ctgaaccatc cagtagccga gagttacgtt tttcctgatt taataaatat ccatatctat
     6181 ttgtctcgtg gtgttttaca gcaactaacc gaaattacta ttttgcatca tccaaccata
     6241 aagagacaac aataattcta gaaaacatat aacaagtgtt tttttctatt tacgtctcat
     6301 acgcaaagtc cactttacca tgttttggaa acttgtaccc aaccattcat ttttcgattt
     6361 tgtaatgagg ctcttttctt ctaacaatca ttgaggtcat tatcatgtgt cctcacctat
     6421 tgtttaaatt gaggcttgcg ctattcccaa aattgtgggt attattttca tatatcagta
     6481 tctgcaattt cggttcaact ccggaagctc ggtacccaac tgtgggaata ctcaggtatc
     6541 gtaagatgca agagttcgaa tctcttagca accattattt tttttctctc aacataacga
     6601 gaacacacag gggcgctatc gcacagaatc aaattcgatg actggaaatt ttttgttaat
     6661 ttcagaggtc gcctgacgca tatacctttt tcaactgaaa aattgggaga aaaaggaaag
     6721 gtgagagcgc cggaaccggc ttttcatata gaatagagaa gcgttcatga ctaaatgctt
     6781 gcatcacaat acttgaagtt gacaatatta tttaaggacc tattgttttt tccaataggt
     6841 ggttagcaat cgtcttactt tctaactttt cttacctttt acatttcagc aatatatata
     6901 tatatatata tatatatttc aaggatatac cattgtaatg tctgccccta agaagatcgt
     6961 cgttttgcca ggtgaccacg ttggtcaaga aatcacagcc gaagccatta aggttcttaa
     7021 agctatttct gatgttcgtt ccaatgtcaa gttcgatttc gaaaatcatt taattggtgg
     7081 tgctgctatc gatgctacag gtgttccact tccagatgag gcgctggaag cctccaagaa
     7141 ggctgatgcc gttttgttag gtgctgtggg tggtcctaaa tggggtaccg gtagtgttag
     7201 acctgaacaa ggtttactaa aaatccgtaa agaacttcaa ttgtacgcca acttaagacc
     7261 atgtaacttt gcatccgact ctcttttaga cttatctcca atcaagccac aatttgctaa
     7321 aggtactgac ttcgttgttg ttagagaatt agtgggaggt atttactttg gtaagagaaa
     7381 ggaagacgat ggtgatggtg tcgcttggga tagtgaacaa tacaccgttc cagaagtgca
     7441 aagaatcaca agaatggccg ctttcatggc cctacaacat gagccaccat tgcctatttg
     7501 gtccttggat aaagctaatg ttttggcctc ttcaagatta tggagaaaaa ctgtggagga
     7561 aaccatcaag aacgaatttc ctacattgaa agttcaacat caattgattg attctgccgc
     7621 catgatccta gttaagaacc caacccacct aaatggtatt ataatcacca gcaacatgtt
     7681 tggtgatatc atctccgatg aagcctccgt tatcccaggc tccttgggtt tgttgccatc
     7741 tgcgtccttg gcctctttgc cagacaagaa caccgcattt ggtttgtacg aaccatgcca
     7801 cggttccgct ccagatttgc caaagaataa ggtcaaccct atcgccacta tcttgtctgc
     7861 tgcaatgatg ttgaaattgt cattgaactt gcctgaagaa ggtaaagcca ttgaagatgc
     7921 agttaaaaag gttttggatg caggtatcag aactggtgat ttaggtggtt ccaacagtac
     7981 caccgaagtc ggtgatgctg tcgccgaaga agttaagaaa atccttgctt aaaaagattc
     8041 tctttttttg tgatatttgt acataaactt tataaatgaa attcataata gaaacgacac
     8101 gaaattacaa aatggaatat gttcataggg tagacgaaac tatatacgca atctacatac
     8161 atttatcaag aaggagaaaa aggaggatgt aaaggaatac aggtaagcaa attgatacta
     8221 atggctcaac gtgataagga aaaagaattg cactttaaca ttaatattga caaggaggag
     8281 ggcatcaca
//
